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Posts Tagged ‘Therapeutic target’


Author: Stephen J. Williams, PhD

     The finding that a substance, derived from vascular endothelium, that could control vascular tone and induce smooth muscle relaxation, led to the discovery of nitric oxide (NO) as a major physiological mediator (1) in many cell types and processes.  Other investigators, working with platelets, determined that nitric oxide is a potent inhibitor, via an autocrine pathway, of platelet aggregation and adhesion to the vessel wall (2).  Nitric oxide is also an important regulator of neurotransmission in the nonadrenergic-noncholinergic system in gastric tissue (3,4).   In addition nitric oxide is involved in macrophage-mediated cytotoxicity, (5)based on the observation the cytotoxic action of macrophages required external arginine, which summarily was converted to citrulline, releasing  the nitric oxide involved in the cell-killing process.  The above physiological responses represent highly regulated, short-term responses that, as seen with classical receptor-based agonists such as epinephrine, terminate once the agonist (NO) is removed.   Given the short half-life of nitric oxide and these rapid physiologic responses, nitric oxide has been given the role of a second messenger within the cell.

However nitric oxide also produces some physiologically, pharmacologically, and pathologically relevant changes, lasting longer time periods, which is the main focus of this article.  For example, nitric oxide is important in the development of long term potentiation (a model of learning and memory), neural plasticity, and neurite outgrowth, revealing nitric oxide can induce more permanent changes in cellular and tissue reorganization (6-9).  Other pathologic and toxicological responses to nitric oxide include cell death from excitotoxic amino acids (glutamate, kainite), oxidative stress, DNA and protein damage, and disease progression in Alzheimer’s disease, epilepsy, aging, apoptosis and Huntington’s chorea (10-12).  These effects persist over longer time frames than the effects which most second messenger systems occur.  These cellular changes can be described by biochemical changes on protein and nucleic acid modification, metabolism (13-15), DNA synthesis and replication, and molecular and organelle reorganization.  The pharmacological and toxicological implications of such cellular changes are inherent in the persistent effects of nitric oxide on biological systems.  The mechanism of nitric oxide-induced physiology and toxicology had been assumed to involve the stimulation of soluble guanylate cyclase, raising intracellular cGMP levels.  As discussed further, this mechanism of action does not account for all the actions of nitric oxide, especially in nitric oxide-induced pathologies.  Other mechanisms of action include post-translational modifications of proteins such as S-nitrosylations, ADP-ribosylations, and a unique nonenzymatic covalent attachment of NAD+ to the regulatory site of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a reaction specific to this dehydrogenase.  GAPDH is a true multifunctional protein involved in diverse cellular functions such as glycolysis, endocytosis, RNA processing and stability, DNA replication and repair, and involved in apoptosis.  GAPDH has been implicated in trinucleotide repeat neurodegenerative disorders such as Huntington’s disease, spinocerebellar ataxia, via binding to the polyglutamated forms of huntingtin and ataxin, protein modifications only seen in these respective diseases. GAPDH has also been implicated in Alzheimer’s disease as well, in genetic linkage studies as well as a β-amyloid precursor protein binding partner (for reviews see (16-20)).

Next to phosphorylation, ADP ribosylation and NAD+ modifications are the second most  common enzymatic  protein modifications in nature and regulates many cellular processes in nervous tissue, tumoral cell growth, cytoskeletal function, cell death and apoptosis, immune function, and bacterial cytotoxicity(21,22). These include poly ADP-ribosylations such as histones in the apoptotic process, and ADP-ribosylation of G-proteins by pertussis and cholera toxin. Interestingly, nitric oxide and other oxidants promote nonenzymatic ribosylation of proteins such as GAPDH.  Unlike the enzymatic reactions, this modification is covalent and generally considered irreversible and either involves nitrosylation of critical reactive cysteine residues or nitric oxide-mediated attachment of the whole NAD+ moiety, a reaction akin to aging of enzymes by reactive oxygen species.  There have been multiple intracellular targets of nitric oxide, with the result of inhibiting activity and/or protein interactions.  These include mitochondrial enzymes such as aconitase (23) and cytochrome oxidase (24), cytosolic enzymes such as cyclooxygenase and affect heme-containing proteins hemoglobin and myoglobin.  Such nitric oxide mediated effects on these systems were cGMP-independent, therefore independent of nitric oxide synthase.  The inhibition of GAPDH glycolytic activity by nitric oxide and NO-mediated NAD+ modification has been widely studied (21,25) and widely accepting to be important in nitric oxide mediated pathology (16,26-33).

So can this NO-NAD+ modification of GAPDH be useful as a therapeutic target for diseases such as Huntington’s, Alzheimer’s or other nitric oxide associated pathologies?   This is as much an intriguing idea as one fraught with caveats and technical issues.   First there is ample evidence that alterations of GAPDH structure/function exist in these neurodegenerative diseases and evidence that this type of modification may be important in the etiology of such diseases(34-41).

Second, as mentioned before, this modification is unique for GAPDH and would offer a disease-specific target(42).  Third, and most interesting, is the multifunctionality of GAPDH, therefore such modification has the possibility for affecting many processes involved in the disease progression.    However there is the big caveat and problem.  Such NO-NAD+ modifications are a covalent reaction, thought to be irreversible.  Studies on purified GAPDH reveal such modification is released by chemicals that can reduce the cysteine covalent bond such as HgCl2 or NaOH treatment(17).  However such treatment would be impractical for in-vivo use.  The ideal situation would be the discovery of an enzyme activity comparable to phosphatases which could enzymatically release the NO-NAD+ modification from GAPDH. A proof of concept experiment could involve creation of a genetically engineered enzyme capable of this reaction.  Therapeutic use of such an enzyme would depend of course on bioavailability.  Interestingly there has been evidence of cellular NO reductase activities, capable of removing the S-nitrosylation on reactive thiols.  Enzymes with denitrosylation activities include the thioredoxin system, superoxide dismutase, and xanthine oxidoreductase (34-40).  Possible therapeutic strategies may include regulation of these intracellular reductase activities.

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Other research paper on Nitric Oxide were published on this Scientific Web site as follows:

Discovery of nitric oxide and its role in vascular biology

Nitric Oxide and Platelet Aggregation

Inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure

Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production

Nitric Oxide in bone metabolism

Nitric oxide and signalling pathways

Rationale of NO use in hypertension and heart failure

Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium

Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function

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